Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are large linear macromolecules composed of covalently-linked nucleotide subunits. DNA is usually found in a "double-stranded" form in which two DNA chains are associated by hydrogen bonding in an antiparallel fashion. RNA usually exists in nature as a single polynucleotide chain. Nucleotides are molecules having a sugar (either deoxyribose or ribose) and a nitrogenous base moiety, and are usually connected together in nucleic acids by a phosphodiester linkage. There are five common nitrogenous bases. Three are found in both DNA and RNA: these are adenine (A), guanine (G) and cytosine (C). The other two, thymine (T) and uracil (U), are specific to DNA and RNA, respectively.
Most (if not all) of every organism's genetic information is transmitted from one generation to the next in the form of DNA or RNA. This information is conveyed in the sequence of the nucleotides along a single nucleic acid chain or "strand", which constitutes a genetic code. Moreover, each of the nitrogenous bases of a nucleic acid strand has the ability to specifically hydrogen bond with one or more other nitrogenous bases of the same or a different nucleic acid strand. Thus, under usual conditions, A hydrogen bonds with T (or U), and C hydrogen bonds with G; this specific hydrogen-bonding is called base-pairing. In double-stranded DNA each of the two strands consists of a chain of nucleotides in which most or all of the nucleotides are base-paired with nucleotides of the other strand. In such a case, the order of nucleotides on one DNA strand determines the order of nucleotides on the other DNA strand. Two nucleic acid strands which are "mirror images" of each other in this way are said to be perfectly complementary.
Nucleic acids are synthesized in vivo by a mechanism exploiting the fact that each nucleic acid strand dictates the order of nucleotides of a perfectly complementary strand; this remains true whether the desired nucleic acid is RNA or DNA, and regardless whether the nucleic acid to be used as a template is RNA or DNA. Most of the specific mechanisms for DNA replication involve the use of a DNA polymerase to sequentially add nucleotides to a 3' hydroxyl group of a polynucteotide primer hydrogen-bonded to the template nucleic acid strand. The newly added nucleotides are chosen by the DNA polymerase based on their ability to base-pair with the corresponding nucleotide of the template strand. This process of adding nucleotides to one end of a primer is sometimes called primer extension.
Unlike DNA synthesis, RNA synthesis does not normally require the existence of a polynucleotide primer. Rather, RNA synthesis is usually mediated by an RNA polymerase which recognizes one or more specific nucleotide sequences of a nucleic acid template. The region of the template to which the RNA polymerase binds, called a promoter, is usually double-stranded. After binding to the promoter, the RNA polymerase "reads" the template strand and synthesizes a covalently-linked polyribonucleotide strand complementary to the template. RNA polymerases from different organisms preferentially recognize different promoter sequences.
DNA and RNA polymerase enzymes have been purified from a number of diverse organisms. Some of these enzymes, such as E. coli DNA polymerase I, the Klenow fragment of DNA polymerase I, and various RNA polymerases are commonly used in vitro as tools in molecular biology and nucleic acid biochemistry research. See generally e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed. Cold Spring Harbor Press 1989).
Another use for nucleic acid polymerases has arisen with the advent of various methods of nucleic acid amplification, such as the polymerase chain reaction (PCR), see e.g., Mullis et al., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159. In the simplest form of PCR, two oligonucleotide primers are synthesized, each primer complementary to a region of a target nucleic acid positioned to the 3' side, with respect to the target nucleic acid, of a target nucleotide sequence region. Each primer is complementary to one of two complementary nucleic acid strands; the target region comprises a nucleotide sequence region encompassing both nucleic acid strands of a double-stranded target nucleic acid. When these primers are allowed to hydrogen-bond ("hybridize") with the substrate and a DNA polymerase is added to the reaction mixture along with nucleotide triphosphates, each hybridized primer is extended by the enzyme in a 5'.fwdarw.3' direction. The reaction mixture is then heated to melt the primer extension product:template hybrid, the temperature is decreased to permit another round of primer/target hybridization, and more DNA polymerase is added to replace the DNA polymerase inactivated by the high temperature step. By repeating the process through a desired number of cycles, the amount of nucleic acids having the target nucleotide sequence is exponentially increased. More recently, a thermostable DNA polymerase derived from Thermus aquaticus has been successfully used in the PCR method to lessen the need for repeated addition of large amounts of expensive enzyme. The Taq polymerase resists inactivation at 90.degree.-95.degree. C., thus obviating the need for repeated additions of enzyme after each round of strand separation.
Other methods of nucleic acid amplification have been devised, such as those using RNA transcription as a step in the amplification process. One such method functions by incorporating a promoter sequence into one of the primers used in the PCR reaction and then, after amplification by the PCR method, using the double-stranded DNA as a template for the transcription of single-stranded RNA by a DNA-directed RNA polymerase, see e.g., Murakawa et al., DNA 7: 287-295 (1988)).
Other amplification methods use multiple cycles of RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, see, e.g., Burg et al,, WO 89/1050; Gingeras et al., WO 88/10315 (sometimes called transcription amplification system or TAS); Kacian and Fultz, EPO Publication No. EPO 408,295 (which enjoys common ownership with the present application); Davey and Malek, EPO Application No. 88113948.9; Malek et al., WO91/02818). These methods make use of an enzyme, reverse transcriptase (RT), which can use RNA or DNA as a template for synthesis of a complementary DNA strand. Some of these methods also utilize cellular RNAse H activity as an essential component. Most retroviral reverse transcriptases, such as those encoded by Moloney Murine Leukemia Virus (MMLV) and Avian Myeloblastosis Virus (AMV), possess an RNA-directed DNA polymerase, a DNA-directed DNA polymerase activity as well as RNaseH activity. RNAse H activity selectively degrades the RNA strand of an RNA:DNA hybrid nucleic acid molecule, thus allowing the amplification reaction to proceed without the need for temperature cycling.
Nucleic acid amplification is an increasingly popular tool for the specific identification and/or amplification of unique or characteristic nucleic acid segments in a variety of settings. Thus, nucleic acid amplification is used in food and agricultural testing, medical diagnostics, human genetic testing and counseling, archeology, and criminal forensics. Because these methods all utilize enzymes, methods of producing, packaging, transporting and storing large quantities of highly active enzymes has become an issue of critical importance in the manufacture, marketing and sale of enzymes and kits for nucleic acid amplification. Specifically, for methods employing transcription-based amplification, commercially acceptable methods and preparations for storing active preparations of reverse transcriptase and RNA polymerase are necessary for the successful manufacture and marketing of kits for nucleic acid amplification.
The usual method of stabilizing reverse transcriptase and RNA polymerase enzymes (as well as many other enzymes used in molecular biology research) is by storing a liquid preparation of each enzyme in a solution containing 50% (v/v) glycerol and a reducing agent such as dithiothreitol (DTT) or .beta.-mercaptoethanol (.beta.ME) at -20.degree. C. This method preserves the activity of the enzymes for many months with little loss of activity. By contrast, enzyme activities are readily lost when the enzymes are stored at room temperature or at 4.degree. C. These preparations are generally shipped from the enzyme supplier to the end user in dry ice; losses of 30% or more of enzyme activity are common during such transport due to freezing and thawing of the enzyme preparation. These enzymes are formulated and supplied separately.
A method of storing and shipping reverse transcriptase and RNA polymerase without the need for refrigeration would obviate the necessity for refrigerated transport and/or methods of cold storage such as dry ice, wet packs, dry packs, or styrofoam shipping containers. Such methods would also be more cost effective, since the production overhead associated with these methods of maintaining enzyme activity would be unnecessary. Methods of storing enzymes which would allow the enzyme preparation to tolerate a limited exposure to higher temperatures would eliminate the losses in enzyme activity which could result if the enzyme preparation sits on a loading dock or in a truck during shipment. Such a method would have to be highly reproducible. Moreover, if the enzymes could be provided in a single container in a form compatible with their intended use (such as in a formulation containing all or most of any necessary co-factors and substrates) such a preparation would be more economical to manufacture and more convenient to use.
Freeze-drying (lyophilization) has been used to preserve foods, biological membranes, whole cells (see. e.g., American Society for Microbiology, Manual of Methods for General Bacteriology 210-217 (1981), and biological macromolecules including enzymes. Lyophilization involves the removal of water from a frozen sample by sublimation under lowered pressure. Sublimation is the process by which a solid is evaporated without passing through the liquid stage.
The theoretical aspects of lyophilization are complex. It is thought that When a biological substance such as a protein is in aqueous solution the molecule is surrounded by a hydration shell comprising water molecules; this hydration shell stabilizes the protein and helps maintain its activity. When water is removed, the protein's reactive groups, which are normally masked by the hydration shell, are free to react with each other, thus forming new, essentially irreversible bonds. These bonds can distort the protein's native conformation. Also, new hydrophobic/hydrophilic interactions may take place in the absence of water which also can distort the conformation of the protein. Since the three-dimensional conformation of many proteins confers a biological activity, the distortion of the conformation can alter biological activities upon drying. By the same mechanism, cross-linking and aggregation of proteins can occur.
Freezing a protein sample prior to drying helps reduce the degree of conformational distortion due to drying. The lowered initial temperature helps keep unwanted reactions between amino acid reactive groups to a minimum by depriving the reactants of energy. At the same time, while in a frozen state the protein has less stearic freedom than when in solution and is less prone to gross conformational change.
However, completely dried lyophilizates tend to have a shorter "shelf" or storage life than do incompletely dried lyophilizates still containing a low percentage of water. Such incompletely dried lyophilizates must often be stored at temperatures no higher than about 4.degree.-10.degree. C., and are still capable of undergoing inactivating chemical reactions that would not be possible were water not present. Thus, while the shelf life of many incompletely dried lyophilized biologically active proteins is longer than those that are completely dried, it is still necessary to refrigerate the preparation in order to maintain activity. Even so, there is a loss of activity in such preparations over a relatively short period of time. Moreover, some enzymes, such as phosphofructokinase, are completely inactivated after lyophilization in the absence of a cryoprotectant, regardless of whether the preparation is completely dried or not. See e.g., Carpenter et al., Cryobiology 25: 372-376 (1988).
As used herein, the term "cryoprotectant" is intended to mean a compound or composition which tends to protect the activity of a biologically active substance during freezing, drying, and/or reconstitution of the dried substance.
The term "stabilizing agent" is meant to mean an agent that, when added to a biologically active material, will prevent or delay the loss of the material's biological activity over time as compared to when the material is stored in the absence of the stabilizing agent.
A variety of cryoprotectant additives have been used or proposed for use as excipients to help preserve biological activity when biological materials, including particular proteins, are dried. Clegg et al., Cryobiology 19: 106-316 (1982) have studied the role of glycerol and/or trehalose in the ability of cysts of the brine shrimp Artemia to remain viable after desiccation. Carpenter et al., Cryobiology 24: 455-464 (1987), report that the disaccharides maltose, sucrose, lactose and trehalose can play a role in increasing the stabilization of phosphofructokinase activity in a purified enzyme preparation subjected to air-drying. EPO Publication No. 0431882A2, discloses a stabilized preparation of purified alkaline phosphatase that had been derivatized and then lyophilized in the presence of mannitol or lactose. EPO Publication No. 0091258A2, discloses a method for stabilizing tumor necrosis factor (TNF) by storage or lyophilization of the purified protein in the presence of a stabilizing protein, such as human serum albumin, gelatin, human .gamma. globulin, or salmon protamine sulfate. U.S. Pat. No. 4,451,569 discloses the use of pentoses, sugar alcohols and some disaccharides to stabilize the activity of purified glutathione peroxidase. The stabilized composition may be freeze-dried and then stored at temperatures below 20.degree. C. EPO Publication No. 0448146A1 discusses stabilized, lyophilized gonadotropin preparations containing a dicarboxylic acid salt. The preparation can further contain a disaccharide such as sucrose or trehalose. Roser, Biopharm, 47-53 (September 1991) discusses preserving the biological activity of various biological molecules dried at ambient temperature using trehalose. PCT Publication No. W087/00196 reports the stabilization of monoclonal antibodies and calf intestine alkaline phosphatase by air drying in the presence of trehalose. PCT Publications W089/00012 and W089/06542 discuss the use of trehalose to preserve some foods and the antigenicity of live virus particles. EPO Publication 02270799A1 reports the stabilization of recombinant .beta.-interferon in a formulation containing a stabilizing agent such as a detergent or glycerol. The compositions can further comprise various sugars including sucrose and trehalose, sugar alcohols, and proteins as additional stabilizing agents; most preferred among these is dextrose.
Some of these additives have been found to extend the shelf life of a biologically active material to many months or more when stored at ambient temperature in an essentially dehydrated form. However, the effectiveness, suitability or superiority of a particular prospective additive depends on the chemical composition of the biologically active material sought to be stabilized; in the case of a protein these factors may include, without limitation, the amino acid sequence of the protein, and its secondary, tertiary and quaternary structure. Thus, whether a particular composition will function to preserve biological activity for a particular biologically active material is not a priori predictable.
Moreover, if a protein is lyophilized, additional factors including, without limitation: the buffer composition, the speed of freezing, the amount of negative pressure, the initial, operating and final lyophilization temperatures and the length of the lyophilization procedure are important in determining the stability and shelf life of the active protein.
Some proteins are known to have multiple enzymatic activities. Thus, retroviral reverse transcriptase enzymes such as those derived from Moloney Murine Leukemia Virus (MMLV-RT) have a DNA-directed DNA polymerase activity, an RNA-directed DNA polymerase activity, and an RNAse H activity. While these activities are contained in the same enzyme, conditions for the preservation of any one of these activities in a dried preparation does not assure that one or both of the remaining enzyme activities will also be preserved under the same conditions.
Moreover, when a particular application requires that the balance of relative specific activities of the three activities of reverse transcriptase remain similar after reconstitution to the balance of these activities before drying, as in the transcription-based nucleic acid amplification system of Kacian & Fultz, supra (which enjoys common ownership with the present application and is incorporated by reference herein), a particular preservation method may upset the delicate balance of these enzymatic activities, thereby making the enzyme unsuitable for such use. Thus, if the RNaseH activity of the enzyme is preserved more than the RNA-directed DNA polymerase activity, the RNA:DNA initiation complex may be degraded before DNA synthesis can begin.
Since a given cryoprotectant composition effective for the long-term preservation of a given enzymatic activity is not clearly effective or superior when applied to another enzymatic activity, different enzymes often require quite different protestants for activity stabilization. As a result, among commercially manufactured lyophilized enzyme preparations, all or most contain only a single enzyme dried in a formulation customized to preserve the activity of that specific enzyme.